Abstract

Axonal loss is the main determinant of permanent neurological disability in the demyelinating disease, multiple sclerosis. However, there remains uncertainty regarding the nature of the cellular factors that elicit axonal injury. Using a non-immune mediated model of demyelination, the Plp1-overexpressing mouse (line #72), it has been shown that early axonal changes occur most frequently in regions of active demyelination where microglia/macrophages are phagocytosing myelin debris (Edgar et al.2010). With this in mind, it seems reasonable to hypothesise that myelin-laden microglia/macrophages are axono-toxic. However, the literature is contradictory, with both pro- (Williams et al., 1994;Mosley and Cuzner, 1996;van der Laan et al., 1996) and anti-inflammatory (Boven et al., 2006;Liu et al., 2006) properties having been ascribed to this population. The aims of this study were therefore to determine, using an in vitro myelinating culture system as a model of CNS white matter, whether myelin-laden microglia/macrophages acquire a (i) pro-inflammatory/axono-toxic or (ii) anti-inflammatory/axono-protective phenotype.
Myelinating cultures were prepared from embryonic mice expressing cyan fluorescent protein under the Thy1 promoter, rendering a subset of neurons fluorescent. Myelin-enriched tissue fractions, to mimic myelin debris, were prepared from wild type or Plp1-overexpressing adult mouse spinal cord and labelled with a rhodamine antibody labelling kit. Myelin fractions were added directly to myelinating cultures at 20, 24 or 27 days in vitro (DIV), for 7, 3 or 1 day(s), respectively. Rhodamine-labelled myelin was phagocytosed by CD45 positive microglia/macrophages within 24 hours of its addition to the cell cultures. The phagocytosis of myelin had no effect on CD45 positive cell density, size or morphology, even after incubation for up to 7 days. Densities of CFP-positive neurites were quantified and the proportion of CFP-positive neurites manifesting signs of injury were calculated. There was no evidence that myelin-laden microglia/macrophages could elicit axonal injury in vitro, with no changes in neuritic density or integrity observed in myelin treated cultures compared to controls. To determine if myelin-laden cells exert an anti-inflammatory, axono-protective effect in an inflammatory environment, cultures were treated with 100 ng/ml lipopolysaccharide (LPS). Pro-inflammatory cytokines, such as IL-1alpha, IL-1beta, IL-6 and TNF-alpha were up-regulated in the medium from the LPS-stimulated cultures. However, these levels were not altered when cultures were pre-treated with myelin debris, to generate myelin-laden microglia. In these LPS-stimulated cultures, neuritic densities and neuritic integrity remained unaltered, either in the presence or absence of myelin-laden microglia/macrophages, compared to controls. Therefore the putative axono-protective effect of these cells could not be determined.